Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy

Mortality of Duchenne Muscular Dystrophy (DMD) is a consequence of progressive wasting of skeletal and cardiac muscle, where dystrophinopathy affects not only muscle fibres but also myogenic cells. Elevated activity of P2X7 receptors and increased store-operated calcium entry have been identified in myoblasts from the mdx mouse model of DMD. Moreover, in immortalized mdx myoblasts, increased metabotropic purinergic receptor response was found. Here, to exclude any potential effects of cell immortalization, we investigated the metabotropic response in primary mdx and wild-type myoblasts. Overall, analyses of receptor transcript and protein levels, antagonist sensitivity, and cellular localization in these primary myoblasts confirmed the previous data from immortalised cells. However, we identified significant differences in the pattern of expression and activity of P2Y receptors and the levels of the “calcium signalling toolkit” proteins between mdx and wild-type myoblasts isolated from different muscles. These results not only extend the earlier findings on the phenotypic effects of dystrophinopathy in undifferentiated muscle but, importantly, also reveal that these changes are muscle type-dependent and endure in isolated cells. This muscle-specific cellular impact of DMD may not be limited to the purinergic abnormality in mice and needs to be taken into consideration in human studies.


Western blot analysis of P2Y receptors and loading controls.
P2Y receptor analyses in primary myoblasts from w/t and mdx animals were performed in cells isolated from 3 individual animals. Duplicate samples containing the same amount of protein were resolved in two gels, transferred and probed simultaneously. This approach was taken because the sizes of some P2Y receptors of interest and proteins used as loading controls (because of their stable expression across cells from the two genotypes and different muscles) overlap in range, and that blot stripping introduces significant errors. Therefore, specific receptors were detected in one blot and tubulin, used as the loading control, was detected in the duplicate blot. Both procedures were done simultaneously.
Western blots of -tubulin used for analyses of P2RY1, P2RY12 and P2RY13.
Blots show -tubulin detected in lysates obtained from myoblasts isolated from TA, GC, SOL and FDB muscles of three w/t and mdx mice. Each lane represents lysate prepared from myoblasts from a specific muscle isolated from one animal. Protein mass standards are also shown as the white ladders.
These blots were exposed to the intensity just before saturation, so the band could be used to confirm the equal protein loading in each well and therefore allow for the estimation of the relative amounts of P2RY1, 12 and 13 receptors.
The identical band intensity across samples demonstrates the suitability of -tubulin as a stable loading control. At the bottom of these membranes the same sets of bands but at a higher intensity as they were used for further calculations are shown again (Fig. 1)   Figure 1.

Figure 2.
Blots show the specific P2Y receptor (indicated) detected in lysates obtained from myoblasts derived from TA, GC, SOL, FDB isolated from three w/t and three mdx mice. Below each blot detecting specific P2Y receptor, the -tubulin obtained as explained in Figure 1 are presented. These were used for the estimation of the relative amounts of the indicated P2Y receptors in myoblasts isolated from specific muscles.

Figure 3
Western blots showing -tubulin detection in samples used for analyses of P2RY2, P2RY4 and P2RY6.
X Wrong sample loaded. This mistake was taken into consideration in further data analyses.

Figure 4
Blots show the specific P2Y receptor (indicated) detected in lysates obtained from myoblasts derived from TA, GC, SOL, FDB isolated from three w/t and three mdx mice. Below each blot detecting specific P2Y receptor, the -tubulin bands are shown obtained as explained in Figure 3 are presented. These were used for the estimation of the relative amounts of the indicated P2Y receptors in myoblasts isolated from specific muscles.
X Wrong sample loaded. This mistake was taken into consideration in further data analyses.

Western blots used for Calcium toolkit proteins detection
In this case, because of a large number of different proteins tested in the same sample, the membranes were cut into strips based on the molecular weights of proteins to be detected. It allowed avoiding membrane stripping, which can introduce additional errors. Moreover, using this approach, fewer animals could be used, as the same lysate was tested for multiple proteins.
-tubulin was used as the loading control using the same approach described above and used to quantify all proteins of interest in samples from a specific muscle.
Each lane represents a protein sample of myoblasts isolated from a specific muscle from an individual mouse.
The left side shows the blot with protein mass markers. The middle is to show the raw data chemiluminescence merged with the mass markers visualised in the visible light (Fusion FX chemidoc). The right side shows blots with specific bands exposed to the intensity before signal